Methods for Sampling and Analyzing Wetland Fungi

Abstract

Most fungi are terrestrial, but representatives of all major groups of fungi along with three groups of fungus-like organisms (water molds, slime molds and lichens), usually studied by mycologists, can be found in wetlands. The primary ecological role of the fungi and water molds in wetland habitats is to decompose dead plant material—both woody and herbaceous debris as well as dead bryophytes. Although sometimes present in wetlands, slime molds and lichens occur almost exclusively on emergent (dry) substrates. Because the vast majority of fungi and fungus-like organisms associated with wetlands are microscopic, efforts to document their distribution and patterns of occurrence often pose a real challenge to ecologists. This chapter reviews some of the more useful and effective methods that can be used to study these organisms in wetland habitats. These include collecting specimens directly in the field, isolating specimens from substrate samples placed in moist chamber cultures and obtaining specimens on various types of organic baits.

Appendices

Student Exercises

3.1.1 Laboratory Exercises

The exercises outlined below provide opportunities to examine the diversity of wetland fungi. Because it has been estimated that 95 % of the world’s fungi have yet to be discovered (Hawksworth 2001) and many of those species that are known can be identified only by experts for the particular group involved, one can anticipate that it will be possible to assign many of the fungi likely to be encountered only to a major taxonomic or ecological group. Detailed field observations provide a basis for developing a better understanding of the effects of various environmental factors on the distribution and abundance of wetland fungi. As such, it is worthwhile to record the temperature of the water when collecting samples. This is particularly important when an effort will be made to isolate and culture the fungi likely to be present. Some fungi tend to grow better at the temperature of the environment from which they were isolated than at room temperature. An effort should be made to identify at least the more abundant plants both surrounding and within the wetland, since they represent the primary sources of plant-derived debris introduced to the wetland itself. If possible, data should be obtained on the physical and chemical characteristics of the water present at a collection site. If the prerequisite equipment is available, such things as levels of dissolved oxygen, pH, and concentrations of nitrate and phosphate should be determined. Having such data allows the conditions present in different wetlands or different portions of the same wetland to be compared.

Hawksworth DL (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycol Res 105:1422–1432

3.1.1.1 Laboratory Exercise #1: Biodiversity of Macrofungi, Slime Molds and Lichens

As already mentioned, the appearance of the fruiting bodies of macrofungi and slime molds in nature is both sporadic and variable through time, whereas lichen thalli are persistent and thus can be collected at any time of the year. As such, any effort to assess the diversity of the first two groups for a particular wetland may not be possible on a given visit. However, when favorable conditions do exist, the “opportunistic sampling method” described above can be used to carry out a survey of fruiting bodies that have developed under natural conditions in the field. In order to quantify the results obtained in such a survey, it is important to utilize a predefined plot size (e.g., 10 by 10 m or 20 by 50 m) and a predefined search time (e.g., 15 min or 1 h) for the area being examined. The type of substrate (e.g., bark, wood, dead leaf or bryophyte) from which each fruiting body was collected and whether the substrate was (a) emergent, (b) on the ground, or (c) submerged should be noted and recorded. If carried out as an educational activity, student participants can be divided into two or more equal numbered groups, with each group establishing at least one replicate plot. For example, two groups of five students could each sample a different wetland plot of the same dimensions in a particular wetland, using the same search time. The results obtained by the two groups could be compared, with the objective of providing answers to a number of questions. For example, how similar were the two plots? What were the differences? Where there any differences in the numbers and types of fungi recovered from different substrates (e.g., wood vs. litter) and the same substrate occurring under different conditions (e.g., aerial woody debris versus submerged woody debris)?

Specimens of macrofungi that have a fleshy texture should be wrapped in sheets of wax paper or aluminum foil, placed in wax paper “sandwich” bags or put in small plastic boxes for temporary storage. Plastic boxes of the type used for fishing tackle are especially appropriate. It is useful to add some type of organic padding (e.g., mosses or pieces of leaves) to the compartment of the box in which a small specimen is to be placed. This prevents the specimen from rolling around and potentially becoming damaged. Wrapped specimens of larger fleshy fungi are best placed in some type of rigid-sided container (bucket, pack basket or pasteboard box) large enough to hold multiple specimens. This protects individual specimens from being crushed while being transported from the field to the laboratory. Specimens of macrofungi with a woody or leathery texture can be placed in small paper bags and, because of their relative toughness, do not have to be handled with as much care.

Paper bags are also appropriate for storing specimens of lichens, while fishing tackle boxes are invariably used for slime molds. Since many of the latter are relatively fragile, providing at least some organic padding for each specimen is strongly recommended. Boxes containing slime molds should be opened immediately upon returning from the field to enable the specimens to air-dry. Otherwise, specimens will be quickly colonized by various filamentous fungi. Dried specimens can be placed in small pasteboard boxes for permanent storage (Stephenson and Stempen 1994). Wet or damp specimens of lichens should be air-dried as soon as possible for the same reason. Simply placing a specimen on a piece of newspaper in the laboratory is usually enough, although drying specimens in a food dehydrator in the same manner as already described for fleshy fungi is sometimes appropriate.

Stephenson SL, Stempen H (1994) Myxomycetes: a handbook of slime molds. Timber Press, Portland

3.1.1.2 Laboratory Exercise #2: Biodiversity of Non-zoosporic Microfungi

The occurrence of various non-zoosporic microfungi associated with wetlands is often difficult to assess, but at least some data on their ecological distribution and relative abundance can be obtained with the use of direct observation with a microscope (either a field microscope or a standard microscope in the laboratory) and various culturing techniques. Two of the most widely used culturing techniques are “baiting” and the use of what are referred to as moist chamber cultures (Fuller and Jaworski 1987; Shearer et al. 2004). Both direct observation and culturing involve collecting samples of substrates that fungi typically colonize in nature. Examples include pieces of semi-submerged or fully submerged woody debris and submerged or emergent portions (dead leaves and stems) of wetland vascular plants such as sedges. Pieces of decorticated woody debris tend to be particularly productive (Fig. 3.9). It is helpful to have either a small knife or a pair of plant cutters to remove small pieces of woody debris from large logs or trees or shrubs still rooted in the water. Samples should be placed in a plastic bag, and a small amount of water added to maintain moist conditions. However, the samples should not be flooded. All samples should be kept cool while being brought to the laboratory. In the laboratory, samples can be examined under a stereomicroscope to check for the presence of fungi, and small amounts of material can be removed with fine-pointed forceps or a dissecting needle and transferred to a glass slide. Once a coverslip has been added, the fungi present in the material on the slide can be checked under a compound microscope. Adding a very small amount of a stain such as methylene blue often causes the vegetative and reproductive structures of fungi to be more easily discernable on the slide. Identification of these fungi is difficult, but notes can be made on any fruiting bodies or other distinctive features observed (e.g., shapes and sizes of conidia). In this way, it is possible to derive at least some estimate as to the diversity of taxa associated with a particular substrate or different types of substrates.

Fig. 3.9

Hyphae and asexual spores of a mitosporic ascomycete (Helicosporium sp.) on a piece of submerged wood

Culturing these same samples (or other samples collected in the same manner) usually yields additional taxa that are not evident through direct observation. The use of moist chamber cultures is a particularly effective way of isolating fungi from all types of substrates. A moist chamber is prepared by first lining the bottom of a Petri dish, glass finger bowl, culture dish or other suitable container with filter paper or a piece of paper towel cut to the appropriate size. Sample material is then placed on the filter paper, a small amount of distilled water is added, and the container set aside for one to several days. If the intent is to maintain the culture for more than 2–3 days, it will be necessary to either place a cover over it (the lid for a Petri dish) or add additional water. The cultures should be examined periodically to note the appearance of various fungi. The same method can be used for slime molds, although cultures usually have to be maintained for a least several weeks to obtain fruitings (Stephenson and Stempen 1994).

A second method of culturing involves attaching a series of “organic baits” (either pieces of woody debris or small-mesh nylon bags containing samples of litter) to a cord anchored to the bottom of a wetland with a brick or a metal stake (Shearer et al. 2004). The organic baits should not be too close to one another, since this may affect the flow of water. One or more of the baits should be retrieved periodically (e.g., once a month or every 2 months for examination). Once they are collected, samples should be placed in plastic bags containing several pieces of paper towels to absorb excess water and stored temporarily in a small cooler away from temperature extremes until taken back to the laboratory.

Fuller MS, Jaworski A (1987) Zoosporic fungi in teaching and research. Southeastern Publishing Corporation, Athens

Shearer CA, Langsam DM, Longcore JE (2004) Fungi in freshwater habitats. In: Mueller GM, Bills GF, Foster MS (eds) Biodiversity of fungi: inventory and monitoring methods. Elsevier Academic Press, Amsterdam, pp 513–531

Stephenson SL, Stempen H (1994) Myxomycetes: a handbook of slime molds. Timber Press, Portland

3.1.1.3 Laboratory Exercise #3: Biodiversity of Zoosporic Fungi

Chytrids and water molds are relatively easy to isolate from samples of water and organic debris that are collected in the field and brought back to the laboratory (Stevens 1974; Shearer et al. 2004). The first step involves collecting a small sample of water with at least some obvious organic debris present. Samples are best collected in a small (25 or 100 ml) screw cap tubes, but most types of containers can be used. Various types of baits are then added to the water in the tubes. Among the most appropriate baits to use are pollen grains, hemp seeds, pieces of snake skin or insect exoskeletons. Once again, samples should be kept away from temperature extremes while being brought back to the laboratory. In the laboratory, the baits in the containers can be examined for the presence of the target organisms in the manner described in the next section.

Shearer CA, Langsam DM, Longcore JE (2004) Fungi in freshwater habitats. In: Mueller GM, Bills GF, Foster MS (eds) Biodiversity of fungi: inventory and monitoring methods. Elsevier Academic Press, Amsterdam, pp 513–531

Stevens RB (ed) (1974) Mycology guidebook. University of Washington Press, Seattle

3.1.2 Classroom Exercises

All of the techniques described thus far can be adapted for classroom activities, since most of these are relatively simple and do not require any specialized equipment. For example, it is possible to have students prepare moist chamber cultures. Each student can prepare one or several moist chamber cultures with a particular type of substrate material or several different types of material, using the same method described above. Students would be expected to check their cultures periodically (at least once a week) and record any fungi that have appeared. Checking the culture under a dissecting microscope is best, but a hand lens can be used if dissecting microscopes are not available. If the students do have access to compound microscopes, they should prepare wet mounts of any filamentous fungus observed. This will allow them to make note of features that are not necessary apparent under the lower magnification available with a dissecting microscope. Various types of information can be complied in this type of activity, including the relative abundance of fungi on different types of substrates (e.g., dead leaves versus woody twigs) and estimates of the numbers of different taxa associated with a particular substrate.

Another possible classroom activity involves “baiting” for chytrids and water molds. Once again, the methods used are the same as already described. A wide range of wetland substrates (surface soil, various types of sediments, woody twigs, and different types of plant debris) in addition to samples of water from the wetland could be investigated, and students can experiment with different types of baits. These baits would be examined periodically for the appearance of chytrids (a compound microscope would be required) or water molds (often readily apparent with the naked eye, but better observed with a hand lens or under a dissecting microscope). The same type of information outlined above should be recorded.

As a variation on this basic activity, different types of substrates (e.g., small fruits and seeds, flower parts and small pieces of leaves from glasses and broad leaf plants) could be collected from the wetland being studied, brought back to a laboratory, autoclaved (to kill any fungi already present) and then used as baits for a series of cultures established from the same sample material. This would allow the students to determine which baits were most effective at attracting zoosporic fungi and whether there were different assemblages of fungi associated with the different types of baits.

Students can gain an appreciation of the morphological diversity of the fungi they have isolated in the laboratory or collected in the field by preparing drawings and detailed descriptions of the fungi in question. Notes should be made of such features as color, shape, and size of any obvious fruiting or vegetative structures as well as the shape and size of any spores that happen to be present.

One important aspect of any classroom activity is having the students become familiar with what is involved in keeping a complete and accurate record of biological research. For example, each student could be expected to prepare a small report on one or more of the fungi observed in a particular activity. This would include (a) where the fungus or sample yielding the fungus was found, including a general description of the wetland involved; (b) when the sample was collected or the fungus actually observed; and (c) how it was collected or isolated (i.e., a description of the techniques used). In addition, the report could include a labelled sketch of the fungus, with diagnostic features pointed out.

3.1.2.1 Classroom Exercise #1: Isolation of Zoosporic Fungi

As noted earlier in this chapter, obtaining chytrids and water molds on baits under laboratory conditions is relatively easy to do and simply involves collecting samples of water, soil, mud or plant debris in the field, bringing these back to the laboratory, placing each sample in a culture dish, glass beaker or other suitable container, adding water (either distilled water or water from the study site) if the sample being examined is soil, mud or plant debris, and then adding baits to the surface of the water. For chytrids, pine (Pinus) pollen grains work exceedingly well, whereas boiled hemp seeds or dead insects are among the most effective baits for water molds.

Another method used to isolate zoosporic fungi directly from field-collected substrates involves placing a small portion of substrate material along with a small amount of water in a Petri dish prepared with water agar or corn meal agar supplemented with streptomycin and penicillin. Dead, but still intact dead leaves, should be rinsed gently with sterile distilled water, cut into small pieces with scissors, and several of these small pieces should be placed on the surface of the agar in a Petri dish. Approximately 0.5 ml of sterile distilled water is then added to each Petri dish, and the dish should then be incubated at 25 C for 24 h. After this period of time, the baits and leaf pieces should be observed under a dissecting microscope with magnification (≥30×). The presence of chytrids and water molds is best determined by carefully examining the margins of the leaf pieces or baits.

3.1.2.2 Classroom Exercise #2: Isolation of Non-Zoosporic Fungi

The methods used to isolate non-zoosporic fungi in the laboratory are essentially extensions of field-based efforts to survey these fungi. Samples of woody debris or dead portions of herbaceous plants, two of the more productive substrates, should be rinsed with sterile tap water or distilled water to remove mud and debris and then used to prepare a series of moist chamber cultures of the type already described. The cultures should be kept at room temperature and under ambient levels of light. It is important to maintain moist conditions in each culture by using a lid to cover Petri dishes, stacking Petri dishes on top of each other, or enclosing the culture in a plastic bag. Samples should be examined for the presence of fruiting bodies under a dissecting microscope (at least 50×) after 1 week and then on a daily basis for a period of up to a month or more. Fruiting bodies of wood-inhabiting ascomycetes may require weeks to develop, whereas reproductive structures and conidia of mitosporic fungi often appear in just a day or two.

Some of the microfungi associated with woody twigs and dead leaves can be induced to form spores under laboratory conditions if the sample material is placed in a conical flask with sterile water or water collected from the study site and then subjected to forced aeration for several days (Tsui et al. 2003). The type of small air pump commonly used with a household aquarium works well for setting up an aeration flask. If some of the bubbles or foam that forms on top of the water in the flask is transferred to a glass slide, a cover slide added, and the slide viewed under a compound light microscope, it is usually possible to observe the spores of various mitosporic ascomycetes and the spore-like propagules produced by aero-aquatic fungi. The latter are especially common in water that is somewhat stagnant. It is important to note that identification in many of these fungi is based largely upon features of their spores and spore-like structures.

Another isolation method that can be used for soil and samples of water involves placing soil particles or a small amount of water directly on the agar surface of a plate prepared with some type of media suitable for the growth of microfungi. The media used vary considerable, but in general, low-nutrient media work best. Among these are cornmeal agar, potato glucose agar and peptone-yeast agars (Stevens 1974). The temperature and light conditions that are most effect will need to be determined through experimentation. Some fungi grow well and produce conidia (usually necessary for identification) under low levels of light, while others appear to require at least some exposure to high light levels. Many of the more common microfungi appear on such media, sometimes in great profusion. To reduce the abundance of fungi appearing in a plate, the sample of water or soil can be diluted. This simply involves thoroughly mixing a small amount of soil in a measured amount (the larger the amount, the greater the dilution) of distilled water in a small tube, adding approximately 0.5 ml of the resulting suspension to a plate and spreading this over the surface of the agar. Colonies of various yeasts appear quickly in these plates, and their sheer abundance is clear evidence of how common these fungi are in wetland and other habitats.

Stevens RB (ed) (1974) Mycology guidebook. University of Washington Press, Seattle

Tsui CKM, Hyde KD, Hodgkiss IJ (2003) Methods for investigating the biodiversity and distribution of freshwater ascomycetes and anamorphic fungi on submerged wood. In: Tsui CKM, Hyde KD (eds) Freshwater mycology. Fungal Diversity Press, Hong Kong, pp 195–209

Literature for Identifying Fungi

As noted earlier in this chapter, identification of most fungi beyond the group to which they belong is difficult, sometimes even for mycologists. Although sources of information do exist, many of these are highly technical. However, there are a number of field guides and similar publications that are relatively non-technical and thus suitable for use by someone without a high level of expertise relating to fungi. These include both general treatments of all fungi (Alexopoulos et al. 1996; Stephenson 2010) as well as publications dealing with specific groups (Lincoff 1981; Fuller and Jaworski 1987; Stephenson and Stempen 1994).

Alexopoulos CJ, Mims CW, Blackwell M (1996) Introductory mycology, 4th edn. Wiley, New York

Fuller MS, Jaworski A (1987) Zoosporic fungi in teaching and research. Southeastern Publishing Corporation, Athens

Lincoff GH (1981) The Audubon Society field guide to North American mushrooms. Alfred A. Knopf, Inc., New York

Stephenson SL (2010) The Kingdom fungi: the biology of mushrooms, molds, and lichens. Timber Press, Portland

Stephenson SL, Stempen H (1994) Myxomycetes: a handbook of slime molds. Timber Press, Portland

Glossary – Wetland Fungi

Agaric:

a type of fleshy fruiting body produced by some macrofungi; it is characterized by a cap with gills (upon which the spores are produced) present on the underside

Bait:

a small piece of organic material, such as a hemp seed, that is placed out in nature or added to a sample of water collected from nature, where it serves as a substrate to isolate an organism of interest (e.g., a chytrid)

Bolete:

a type of fleshy fruiting body produced by some macrofungi that is characterized by a cap that contains many small tubes on the underside within which the spores are formed

Bryophilous:

either living on or producing fruiting bodies in association with bryophytes

Cap:

the portion of a fleshy fruiting body produced by some macrofungi that sits on top of the stalk and contains the spore producing region; a more technical term is “pileus”

Chitin:

a nitrogen-containing polymer that serves as the primary structural component of the cell wall of fungi; also found in the exoskeletons of arthropods

Coenocytic:

a cell that contains multiple nuclei

Conidium (plural: conidia):

asexual spores produced by non-zoosporic microfungi

Ectomycorrhizal:

a mutually beneficial relationship that develops between a fungus (typically a basidiomycete) and the root cells of a living plant; the hyphae of the fungus do not penetrate the root cells

Endomycorrhizal:

a mutually beneficial relationship that develops between a fungus (typically a glomeromycete) and the root cells of a living plant; the hyphae of the fungus do penetrate some of the root cells

Endophyte:

a fungus that lives inside the tissues of a living plant

Epiphyte:

a fungus that lives on or produces fruiting bodies on the surface of a living plant

Eukaryotic:

an organism made up of cells with a nucleus

Fruiting body:

a reproductive structure produced by fungi; the spores are produced within or on the surface of this structure

Germination:

the process by which the initial hypha of a fungus emerges from a spore

Heterotroph:

an organism that cannot manufacture its own food and instead derives its food from some outside source, either dead organic matter or a living organism; all fungi are heterotrophs

Hypha (plural: hyphae):

one of the microscopic, thread-like filaments making up the body of a fungus

Life cycle:

the series of developmental changes through which a fungus passes from its inception (usually as a germinated spore) to the mature state in which a fruiting structure is produced

Lignin:

a structural compound associated with plant cell walls in woody tissues

Macrofungus (plural: macrofungi):

a fungus that produce fruiting bodies (mostly above ground) that are fleshy and large enough to be noticed by a casual observer in the field

Microfungus (plural: microfungi):

a fungus that produce fruiting bodies that are small and inconspicuous and therefore not easily detected in the field

Moist chamber culture:

a simple isolation technique in which a substrate sample is brought into the lab, placed in a container and kept moist in order to observe the organisms that develop

Mushroom:

a common name often used to describe the type of fruiting body produced by some macrofungi

Mutualistic:

an association between two organisms in which both members benefit

Mycelium (plural: mycelia):

a collection of interwoven hyphae making up the body of the fungus

Mycology:

the scientific study of fungi

Mycologist:

a person who studies fungi

Parasite:

an association in which an organism derives its food from a second organism (the host), usually without killing the latter

Pathogen:

an association in which an organism derives its food from a second organism (the host), ultimately killing the latter

Polypore:

a type of tough and often resistant fruiting body produced by some macrofungi in which the spores develop within tiny tubes, thus giving the spore-bearing surface a porous appearance

Puffball:

a type of more-or-less globose fruiting body formed by some macrofungi in which the spores are produced internally in a large mass; the spores are released when the outer wall ruptures in some fashion

Saprotroph:

an organism that obtains its food from dead organic matter

Septum (plural: septa):

a cross-wall (often with a tiny opening or pore) that forms inside the hyphae of many fungi, dividing a hypha into cell-like units

Spore:

the reproductive propagule of a fungus

Spore print:

a technique used to help in the identification of some macrofungi by providing samples of spores for microscopic examination and allowing the investigator to determine the color of the spores in mass; a spore print is obtained by placing a portion of a relatively fresh fruiting body on a piece of paper and allowing the spores to be deposited

Substrate:

the substance or material upon which a fungus lives, feeds or produces its fruiting bodies

Zoospore:

a motile spore that is capable of moving through its environment by means a flagellum; produced by chytrids and the fungus-like water molds